The present invention relates to compositions for inducing protective antibodies against hepatitis. It also relates to a vector comprising a nucleotide sequence coding for at least a portion of a virus protein, which is capable of being expressed in muscle cells. In addition, the invention relates to compositions capable of inducing a T cell response in chronic HBV carriers.
Hepatitis B is a widespread and serious international health problem. In addition to causing acute hepatitis and liver damage, the hepatitis B virus (HBV) can cause cirrhosis and hepatocellular carcinoma (Davis, Hum. Molec. Genet. 2:1847-1851 (1993)).
The HBV is a 42-nm particle (Dane particle) consisting of a lipoprotein envelope enclosing a core protein (capsid) and the viral genome, which contains only four genes (S, C, P, X). The major (or small) envelope protein, which includes the surface antigen of HBV (HBsAG) is encoded by the S gene and is organized into dimers of one glycosylated and one unglycosylated polypeptide (Petersen, J. Biol. Chem. 256:6975-6983 (1981)). Present in smaller amounts are the middle and large envelope proteins, which are encoded by the pre-S2 and S or pre-S1, pre-S2 and S genes, respectively. The predominant form of HBsAg secreted by infected cells is not the Dane particle, however, but 22-nm particles or filaments, which are empty viral envelopes composed solely or predominantly of major (small) envelope protein and sometimes small amounts of middle and large proteins (Maupas, Lancet 1:1367-1370 (1976)). The 22-nm particles are seen to persist in the plasma of chronic carriers (Davis, 1993).
In 1976, the first vaccine against HBV comprising 22-nm HBsAg particles was applied to humans (Maupas, 1976). The particles were purified from the plasma of chronic carriers and treated to eliminate possible co-purified infectious HBV or other pathogens. While this vaccine was effective, mass immunization was not feasible due to the long and expensive purification procedure, the need to assay each batch on chimpanzees for safety, and the limited supply of chronically infected human plasma (Maugh, Science 210:760-762 (1980); Stephenne, Vaccine 6:299-303 (1988)).
The present vaccines are produced employing genetic engineering techniques to create HBsAg-producing cell lines. One frequently used vaccine is a second generation vaccine based on recombinant yeast cells containing the S gene for HBsAg (Valenzuela, Nature 298:347-350 (1982)). Another vaccine commonly used in France is a third generation vaccine based on a line of Chinese hamster ovary cells containing both the S and pre-S2 genes (Michel, Proc. Natl. Acad. Sci. USA 81:7708-7712 (1984)). While the present protein vaccines are highly effective and safe, the production and maintenance of these vaccines is time-consuming and expensive (Davis, 1993). On the other hand, the production of a viral vaccine is not feasible due to safety considerations.
Immunization by DNA-based vaccines has been the object of several studies since the beginning of the 1990s. A DNA-based vaccine involves the transfer of a gene or at least a portion of a gene, by direct or indirect means, such that the protein subsequently produced acts as an antigen and induces a humoral- and/or cellular-mediated immunological response.
Ulmer et al. (Science, 259:1745-1749 (1993)) obtained protection against the influenza virus by induction of the cytotoxic T lymphocytes through injection of a plasmid coding for the influenza A nucleoprotein into the quadriceps of mice. The plasmid used carries either the Rous sarcoma virus promoter or the cytomegalo virus promoter.
Raz et al. (Proc. Natl. Acad. Sci. USA 90:4523-4527, (1993)) injected vectors comprising the Rous sarcoma virus promoter and a gene coding for interleukin-2, interleukin-4, or the xcex21-type transforming growth factor (TGF-xcex2 1). The humoral and cell-mediated immune responses of the mice to which these plasmids have been intramuscularly administered are improved.
Wang et al. (Proc. Natl. Acad. Sci. USA, 90:4156-4160, (1993)) injected a plasmid carrying a gene coding for the envelope protein of the HIV-1 virus into mice muscles. The plasmid injection was preceded by treatment with bupivacaine in the same area of the muscle. The authors demonstrated the presence of antibodies capable of neutralizing the HIV-1 virus infection. However, the DNA was injected twice a week for a total of four injections.
Davis et al. (Compte-Rendu du 28 xc3xa8me Congrxc3xa8s Europxc3xa9en sur le muscle, Bielefeld, Germany, Sep. 21-25 1992) injected plasmids carrying a luciferase or xcex2-galactosidase gene by pretreating the muscles with sucrose or a cardiotoxin. The authors observed the expression of luciferase or xcex2-galactosidase.
More recently, an article published in Science et Avenir (September 1993, pages 22-25) indicates that Whalen and Davis succeeded in immunizing mice against the hepatitis B virus by injecting pure DNA from the virus into their muscles. An initial injection of snake venom toxin, followed 5 to 10 days later by a DNA injection, is generally described. However, the authors specify that this method is not practical.
These studies were preceded by other experiments in which various DNAs were injected, in particular into muscle tissues. For example, the International application, PCT/US90/01515 (published under No. WO-90/11 092), discloses various plasmid constructions, which can be injected in particular into muscle tissues for the treatment of muscular dystrophy. However, this document specifies that DNA is preferentially injected in liposomes.
Additionally, Canadian patent CA-362.966 30 (published under No. 1,169,793) discloses the intramuscular injection of liposomes containing DNA coding, in particular, for HBs and HBc antigens. The results described in this patent mention the HBs antigen expression. The presence of anti-HBs antibodies was not investigated.
International application PCT/FR92/00898 (published under No. WO-93/06223) discloses viral vectors, which can be conveyed to target cells by blood. These vectors are recognized by the cell receptors, such as the muscle cells, and can be used in the treatment of muscular dystrophy or of thrombosis.
The DNA-based vaccines suggested by the prior art have not been capable of practical uses. For example, some bare DNA used to vaccinate the mice was pure DNA from the virus. This type of treatment can not be considered for human vaccination due to the safety risks involved for the patients. Additionally, earlier experiments in which the injected DNA is contained in liposomes did not exhibit an immune response.
The present inventors have succeeded in developing effective DNA-based immunizing compositions capable of inducing immune responses against infectious viruses without the detrimental effects on human health.
The present invention relates to a composition capable of inducing T cell response, and more particularly, a cytotoxic response comprising a nucleotide sequence expressed in muscle cells. The nucleotide sequence comprises a gene or complementary DNA coding for at least a portion of a virus protein and a promoter allowing for the expression of the gene or complementary DNA in the muscle cells.
The invention further relates to the vector, which serves as a vehicle for the gene or complementary DNA coding for at least a portion of a virus protein and a promoter allowing for the expression of the gene or cDNA, which is administered to an individual to be immunized.
In addition, the inventors have developed a non-lipid pharmaceutical composition comprising at least, on the one hand, a substance capable of inducing a coagulating necrosis of the muscle fibers, such as bupivacaine, and, on the other hand, a vector including the gene or complementary DNA coding for at least a portion of a virus protein, which is expressed in muscle cells, and the promoter. Preferably, the substance capable of inducing a coagulating necrosis of the muscle fibers is first administered into the muscle of an individual to be immunized. Then, at least five days later, the vector is administered into substantially the same location of the individual""s muscle.
The inventors discovered that the compositions of the instant invention are capable of breaking T-cell tolerance to HBsAg in a mouse model for chronic HBV carriers. Thus, the present invention is further directed to the treatment of chronic HBV carriers.